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COX-2

COX-1 and COX-2, also called PGHS-1 and PGHS-2, regulate a key step in prostaglandin and thromboxane synthesis and are the targets of nonsteroidal antiinflammatory drugs (NSAIDs). Prostaglandins are implicated in various pathophysiological processes such as inflammatory reactions, gastrointestinal cytoprotection, hemostasis and thrombosis, as well as renal hemodynamics. Whereas COX-1 presents a widespread constitutive expression, COX-2 is undetectable in most normal tissues (except for the central nervous system, kidneys, and seminal vesicles), but is induced by various inflammatory and mitogenic stimuli. More recently, a third isoform named COX-3 was identified as a COX-1 splicing variant. This new isoform may play a role in processes such as fever and pain. Additionally, a high level of COX-2 expression is found usually in cancer cells. For example, COX-2 overexpression is related to poor prognosis in certain breast cancers and endometrial adenocarcinomas.

Patho-physiological Function
COX-2, unlike COX-1, is induced in inflammatory cells when they are activated by various inflammatory and mitogenic stimuli in order to produce the prostanoid mediators that trigger important inflammatory processes including physiological and pathological situations. Although inflammation is initially a necessary process to fight infection or build up an efficacious inmmune response, when it is maintained or remains uncontrolled it can provoke chronic pathologies and tissue damage. This is why the inhibitios of COX proteins have created considerable interest as potentially potent anti-inflammatory targets what has led to the development of the "coxibs". However, COX-2-produced prostanoids also regulate many important physiological functions such as vascular, bronchial, or gastrointestinal contractility by regulating smooth muscle tone, uterine contractility during labor, and the activity of hormones and fat metabolism among other functions.

This fact makes it difficult to design pure anti-inflamatory drugs based on COX inhibition in the absence of side effects. For instance, both COX-1 and COX-2 help to convert essential fatty acids to other prostanoids that actually reduce inflammation or serve other regulatory functions. Also, the excess arachidonate that cannot be converted into prostaglandins upon COX inhibition can be derived into leukotriene synthesis thus sustaining allergic reactions, or alternatively into thromboxanes, which may be responsible for the increased clotting and subsequent heart attacks detected with the use of some COX-2 inhibitors (celecoxib, rofecoxib, ...).

Constitutive levels of COX-2 are generally low in most tissues, although there are some significant exceptions. For example, there is a considerable pool of “constitutive” COX-2 present in the central nervous system (CNS) and some other tissues, although its function is not yet completely clear.

Moreover, COX-1, that is present ubiquitously, has a “housekeeping” role in the body, being involved in tissue homeostasis, and it appears to be responsible for the production of the prostaglandins involved in gastric cytoprotection, platelet aggregation, renal blood flow autoregulation and the initiation of parturition.

Structure
 In 1994, Picot et al published the first three-dimensional (3D) structure of a COX enzyme, the ovine COX-1 complexed with the NSAID flurbiprofen. Soon afterward, the crystal structures of human and murine COX-2 followed. First, the three-dimensional structure of human COX-2 was assessed by means of sequence homology modeling, but in 1996, Luong, C. et al and Kurumbail, R.G et al published two crystal structures of the recombinant human and mouse COX-2 isozymes, respectively, complexed with different selective inhibitors. Given its pharmacological importance as a therepeutic target, drug interactions with COX were one of the first issues to be addressed, and complexes containing a number of different NSAIDs have been studied crystallographically. The structural analysis of COX complexed with substrates or products was more difficult to pursue for a number of technical reasons. However, within the past years, crystal structures of murine COX-2 complexed with AA and EPA have also been determined.

PGHSs are bifunctional homodimers. Both COX-1 and COX-2 are membrane-bound enzymes and are present on the luminal surfaces of the endoplasmic reticulum and of the inner and outer membranes of the nuclear envelope. However, recently, using cultured endothelial cells and fibroblasts a fraction of COX-2 protein was shown to be localized to plasma membrane in caveolae-like structures. The primary structure of nascent COX-2 is of 604 amino acids and it is processed into a mature form by removal of signal peptides giving a protein of 587 amino acids. PGHS-2 is variably glycosylated at two to four sites, leading to the formation of doublets or sometimes triplets that can be detected on SDS-PAGE. In murine PGHS-2 carbohydrate moieties are linked to Asn-68, Asn-144, and Asn-410 in each monomer.

The COX monomer consists of three structural domains : the N-terminal EGF domain, a membrane binding domain (MBD) and a large C-terminal globular catalytic domain containing the heme binding site. The C-terminal segments beyond Pro583 (35 amino acids in COX-2) have not been resolved crystallographically. Collectively, these domains are made up of 25 alpha helices, seven 310 helices , four beta sheets as well as five disulfide bonds which contribute to the interface binding of the two individual monomers to complete the enzyme.

Epidermal Growth Factor Domain
The EGF and catalytic domains create the subunit interface in the dimer and place the two MBDs in a homodimer about 25 amstrongs apart. The EGF domains create a substantial portion of the dimer interface. EGF domains are common in several families of membrane and secreted proteins. Typically, the EGF domain occurs at a position in the primary sequence N-terminal to a membrane anchor, such that these domains always occur on the extracytoplasmic face of the membrane. Some authors have suggested that the EGF domains may play a role in the insertion of COX into the lipid bilayer.

Membrane Binding Domain
PGHS-2 associate with only one face of the membrane bilayer through a monotopic membrane binding domain (MBD) that is comprised of four short, consecutive, amphipathic α-helices (helices A–D) that include residues 59-111 in human PGHS-2. Three of the four helices lie roughly in the same plane while the last helix angles “upward” into the catalytic domain. Hydrophobic and aromatic residues protrude from these helices to create a hydrophobic surface that would interact with only one face of the lipid bilayer.

Catalytic Domain
The catalytic domain comprises the bulk of the COX monomer and is almost entirely composed of α-helical secondary structure. As mentioned before COX are bifunctional proteins so two types of reactions can be differentiated: the heme-dependent bis-oxygenase or COX reaction that converts AA to PGG2 and the subsequent peroxidase (POX) reaction that reduces the 15-hydroperoxide of PGG2 to form PGH2.

Peroxidase Active Site Structure
The POX active site is in a large groove on the side opposite of the MBD. The structures of the peroxidase active sites of PGHSs are similar to those of other heme peroxidases. This site includes a heme group and the iron (III) in the center of this heme is coordinated by His-388 and by His-207.

Heme-dependent peroxidase activity is implicated in the formation of a proposed Tyr-385 radical, which is required for cyclooxygenase activity. Gln203 is also important in catalysis, although its function has not been resolved. Mutations of Gln203, His207, or His388 lead to a reduction or elimination of peroxidase activity.

The COXs bind 1 mole of ferric-protoporphyrin IX per mole monomer for full activity, as expected for a heme-dependent peroxidase.

Cyclooxygenase Active Site Structure
PGHS-1 and 2 monomers each contain a 25-°A hydrophobic channel that originates at the membrane binding domain and extends into the core of the globular domain. The MBD forms the entrance and the first half of the channel and allows arachidonate and O2 to enter directly from the apolar compartment of the lipid bilayer. Several amino acids composing the upper half of the channel are uniquely important to cyclooxygenase catalysis. Twenty-four residues line the hydrophobic cyclooxygenase active site with only one difference between the isozymes—Ile at position 523 in PGHS-1 and Val at position 523 in PGHS-2. Amino acids lining the hydrophobic cyclooxygenase active site channel include Leu117, Arg120, Phe205, Phe209, Val344, Ile345, Tyr348, Val349, Leu352, Ser353, Tyr355, Leu359, Phe381, Leu384, Tyr385, Trp387, Phe518, Ile/Val523, Gly526, Ala527, Ser530, Leu531, Gly533, Leu534. Only three of the channel residues are polar (Arg120, Ser353, and Ser530).Tyr 385 in its radical form is the responsible for abstracting a proton from arachidonic acid during its conversion to PGG2.Ser530 is the site of acetylation by aspirin and Arg120, which is positioned about midway between the entrance and the apex of the active site , binds to the carboxylate groups of fatty acids and many NSAIDs. 

NSAIDs


Non-steroid anti-inflammatory drugs are a chemically heterogeneous group of compounds whose major function is the inhibition of cyclooxygenases (Table 1). Apart from their anti-inflammatory effect, they also present analgesic and antipyretic properties.

Classical NSAIDs, as salicylate or phenoprofen, are mostly inhibitors of both isoenzymes, although each isoform is inhibited in a different level (Table 2). Chronic users of NSAIDs develop gastric ulcers or gastrointestinal complications, explained by the inhibition of COX-1. For this reason, selective inhibitors of COX-2, as celecoxib, valdecoxib and etoricoxib, have been developed. They don’t cause gastric pathology, but they has been proven to be responsible of nephrotoxicity in some patients.

The majority of NSAIDs inhibit competitively the initial dioxygenation. In general, these drugs block COX-1 in a quicker manner, whereas COX-2 inhibition is a more time-dependant event, and usually irreversible. The new COX-2 inhibitors exhibit PGHS-2 selectivity because they inhibit this isoform by a time-dependent, pseudoirreversible mechanism, whereas they inhibit PGHS-1 by a rapid, competitive, and reversible mechanism. The inhibition mechanism consists of the entrance of the drug by the hydrophobic channel and the formation of hydrogen bonds with Arg120. This interaction prevents the fatty acids from entering the catalytic site. Selectivity of COX-2 inhibitors is mainly mediated by the substitution of Ile523 in COX-1 with Val523 in COX-2, which results in the presence of a small side pocket adjacent to the active site channel, appreciably increasing the volume of the COX-2 active site. The effect of this change is compounded by the substitution of Val434 in COX-2 for Ile434 in COX-1 within the second group of amino acids conforming the active site. The combination of these two substitutions in COX-2 allows a neighboring amino acid, Phe518, to swing out of the way, which further increases access to the side pocket.

In addition, other NSAIDs present alternative inhibition mechanisms. Acetylsalicylic acid, for example, makes its function by irreversible acetylation of COX-2 in Ser516.

Finally, paracetamol is considered an atypical NSAIDs, not only because of its lack of anti-inflammatory properties but also because it does not interact neither with COX-1 nor with COX-2. It has been proposed that paracetamol may act as an analgesic and antipyretic drug by inhibition of COX-3.

Regulation
COX-2 overexpression is a very important process since it has significant tissue-specific consequences and is associated with inflammatory diseases, cancers and term/preterm labour, thus making COX-2 an important target for pharmacological intervention.

The expression of COX-2 in many specialized cell types appears to be differentially sensitive to the different stimuli that regulate the unique physiological activities of each tissue.

This physiological regulation can be produced at various levels :

-	Transcriptional regulation

-	Post-transcriptional regulation: via 3’UTR, miRNAs (microRNAs) and alternative polyadenylation

Transcriptional regulation
Transcriptional activation of COX-2 occurs quickly and transiently in response to different stimuli, for example: pathogens, cytokines, nitric oxide, irradiation, growth factors and various extracellular ligands.

The 5-UTR (untranslated region) of the COX-2 gene has several transcription factor response elements, including two NF-κB (nuclear factor κB) motifs, two AP-1 (activator protein 1) sites and two CREs (cAMP-response elements), among others. Transcriptional regulation of COX-2 may also be physically influenced by chromatin remodelling events such as changes in acetylation status of histones and non-histone proteins. For example, the acetylation of NF-κB components by the transcriptional coativator p300 (histone acetyltransferase [HAT]) can activate the COX-2 expression, while the hypermethylation of the CpG islands results in transcriptional silencing. It is also known that the histone deacetylase inhibitors (iHDAC) suppress the activation of the expression in human primary myometrial cells and in cancer cell lines, by preventing the binding of the transcription factor, c-Jun, to the COX-2 promoter.

Via 3’-UTR
The 3’-UTR of COX-2 is a complex region that contains multiple copies of AREs (AU-rich elements) throughout sequence, which, when bound by specific trans-acting ARE-binding factors, influence COX-2 mRNA stability and also translational efficiency. A lot of studies have introduced a new model to the gene regulation of COX-2 by investigating the combined contribution of both transcription and mRNA stability events. For example, one group has reported that the binding of the protein CUGBP2 (CUG triplet repeat, RNA-binding protein 2) in specific AREs within the first 60 nucleotides of the COX-2 3’-UTR can stabilize the COX-2 mRNA inhibiting its translation. Also, there is evidence that mitogenic inhibitors (e.g. taxanes) can control COX-2 transcription via PKC (Protein Kinase C)-p38 MAPK (Mitogen-Activated Protein Kinase) signaling cascade and it is known that the stability of COX-2 mRNA can be controlled by the binding of HuR (a mRNA-stabilizing factor) to AREs in 3’-UTR of COX-2.

miRNAs (microRNAs)
A recent study has demonstrated that the microRNAs miR-101a and miRNA-199a can interact with the COX-2 3’-UTR in vitro thus repressing its translation.

Alternative polyadenylation
The human COX-2 3’-UTR has several polyadenylation sites. COX-2 uses two alternative polyadenylation sites, in a tissue-specific manner, which derives in the formation of 2 COX-2 mRNAs: one with 2.8 kb and another one with 4.6 kb. It is known that selection of the proximal polyadenylation signal is enhanced by presence of additional USEs (Upstream Sequence Elements) where four RNA-binding proteins (U1A, PTB, p54nrb and PSF) can bind, enhancing the recruitment and stabilization of core adenylation factors on the COX-2 mRNA.